B CELLS AND ANTIBODY RESPONSE

R. Motallebzadeh¹, S. Rehakova¹, M. Negus², M. Goddard³, I. Harper⁴, S. Sivaganesh², E.M. Bolton², C.J. Callaghan⁴, J.A. Bradley⁵, N. Ruddle⁶, G.J. Pettigrew^4
1Surgery, Cambridge University, Cambridge/UNITED KINGDOM, ²Department Of Surgery, University of Cambridge, Cambridge/UNITED KINGDOM, ³Department Of Pathology, Papworth Hospital, Cambridge/UNITED KINGDOM, ⁴Department Of Surgery, Addenbrooke's Hospital, University of Cambridge, Cambridge/UNITED KINGDOM, ⁵Transplant Surgery, Addenbrooke's Hospital NHS Trust, Cambridge/UNITED KINGDOM, ⁶, Yale University, New Haven/CT/UNITED STATES OF AMERICA

Body: Introduction Tertiary lymphoid organs (TLOs) and intra-graft lymphatic vessel (LV) proliferation have been described in allografts and may contribute to rejection. The lymphotoxin-β receptor (LTβR) signalling pathway is essential for lymph node development in ontogeny and vascular endothelial growth factor receptor (VEGFR-3) signalling is required for lymphangiogenesis. Here we study the effect of blocking these pathways on TLO and LV formation in a model of allograft vasculopathy (AV).

Methods
The presence of TLOs in day 50 bm12 heart allografts in B6 recipients was confirmed by: discrete aggregates of B and T cells associated with high endothelial venules. LV density was assessed by staining with anti-LYVE-1 mAb. LTβR signalling was blocked by weekly intra-peritoneal injection of 100μg LTβR-Ig fusion protein (n=5), and VEGFR-3 signalling by injection of 25mg/g mF4-31C1 mAb three times per week for 3 weeks. Control recipients received rat IgG (n=5). Donor T cells within bm12 heart allografts provoke, in B6 recipients, anti-nuclear autoantibody (Win TS 2009); this was quantified by binding test sera to nuclear antigen expressing HEp-2 cells and by anti-ds DNA ELISA.

Results
All bm12 heart allografts from control-Ig treated B6 recipients contained TLOs, composed predominantly of B cells. Although LV density was reduced in recipients treated with mF4-31C1 (1884.6μm² vs control 3368μm², p=0.03), TLO formation was unaltered. In contrast, treatment with LTβR-Ig resulted in fewer TLOs (median/heart=0 vs 2, p=0.01), less dense LV (1427.4 μm², p=0.008), and a non-significant reduction in severity of AV. However, autoantibody responses were significantly diminished (figure 1). To address whether the reduction in autoantibody following LTβR-Ig treatment is due predominantly to blocking TLO formation (rather than influencing signalling in conventional lymphoid tissue), a further group of B6 mice were challenged with bm12 CD4 T cells. The autoantibody response that this provoked was reduced by LTβR-Ig treatment but much less so than in heart-grafted mice.

Conclusions
Blocking VEGFR-3 signalling prevented LV, but not TLO, formation within allografts. In contrast, LTβR-Ig treatment blocked TLO development and was associated with a reduction in autoantibody. Although LTβR-Ig treatment influences responses within conventional lymphoid tissue, our results suggest that this reduction was due predominantly to the prevention of TLO formation and confirm that the lymphoid microenvironment of the allograft plays an important role in chronic rejection.

Disclosure: All authors have declared no conflicts of interest.